The use of ULVs as drug carriers has been the subject of extensive investigation. The lack of stability of lecithin liposomes in vivo and the toxicity of many of the lipid compounds added to increase stability in vivo have limited the usefulness of liposomes as carriers for administration of drugs. The proteoliposome is an alternative vehicle for these objectives.
Studies of the properties of proteins that are integral components of membranes depend on dissolution of the membrane with detergents, separation of the protein of interest from other membrane components, and finally re-introducing the protein into a matrix of phospholipid. The latter may have a variety of physical structures, depending on the complex of protein and phospholipid being studied. The complex of most interest, because of its resemblance to naturally occurring biological membranes, is reconstitution of integral membrane proteins into unilamellar bilayers of phospholipid. Protein-phospholipid structures of this form can be obtained in several different ways. As far as is known, the method for preparing these structures does not affect the properties of the protein or lipid, assuming the protein functions in a stable manner under the conditions used to reconstitute the protein-phospholipid vesicle.
The most popular techniques for achieving reconstitution of a protein-phospholipid vesicle include co-sonication of protein and phospholipid (Racker, E. (1973) Biochem. Biophys. Res. Communs. 55, 224-230; Carol, R. C. and Racker, E. (1977) J. Biol. Chem. 252, 6981-6990; Banerjee, R. K., Shertzer, H. G., Kanner, B. I. and Racker , R. (1977) Biochem. Biophys. Res. Communs. 75, 772-778) and slow dialysis of mixtures of integral membrane protein, phospholipid and detergent (Kagawa, Y. and Racker, E. (1971) J. Biol. Chem. 246, 5477-5487; Hinkle, R. C., Kim, J. J. and Racker, E. (1972) J. Biol. Chem. 247, 1338-1339; Racker, E. (1972) J. Biol. Chem. 247, 8198-8200; Kagawa, Y., Kandrach, A. and Racker, E. (1973) J. Biol. Chem. 248, 676-684). Direct insertion of selected small proteins, such as mellitin (Vogel, H. (1981) FEBS. Lett. 134, 37-42; Jahnig, F. (1983) Proc. Nat'l Acad. Sci. 80, 3691-3695; Vogel, H., Jahnig, F., Hoffman, V. and Stumpfel, J. (1983) Biochem. Biophys. Acad. 733, 201-209) or microsomal cytochrome b.sub.5 (Enoch, H. G., Fleming, P. J. and Strittmatter, P. (1977) J. Biol. Chem. 252, 5656-5660) into preformed phospholipid vesicles apparently can be achieved. In a limited number of instances, the use of specific mixtures of phospholipids in the bilayer has led to direct and spontaneous incorporation into the bilayer of specific integral membrane proteins, as for example cytochrome oxidase (Eytan, G. D., Matheson, M. J. and Racker, E. (1976) J. Biol. Chem. 251, 6831-6837; Eytan, G. D. and Broza, R. (1978) J. Biol. Chem. 253, 3196-3202). Small proteins, such as mellitin, that insert spontaneously into vesicles appear to attach to the membrane by a single region of .alpha.-helix and the driving force for insertion is believed to be a transition from the form of a random coil in water to that of an .alpha.-helix in the bilayer (Jahnig, F. (1983) Proc. Nat'l Acad. Sci. 80, 3691-3695). The mechanism by which a protein such as cytochrome oxidase incorporates spontaneously into bilayers with limited specific composition is unclear.
The most generally useful method for reconstituting large integral membrane proteins, based on its popularity, is the formation of protein-lipid vesicles by slow dialysis of a mixture of protein, phospholipid and detergent. This and the method of co-sonication have been used almost exclusively as a means for studying the influence of the lipid millieu on the properties of the protein. These techniques have proved extremely valuable in this context. On the other hand, these commonly used techniques for reconstituting complexes of protein and lipid provide little information on the mechanism(s) by which integral membrane proteins in cells might enter biological membranes. Thus, although many proteins insert into intracellular membranes as they are translated on ribosomes attached to these membranes (Blobel and Doberstein (1975) J. Cell. Biol. 67, 835-851), it is clear that this is not so for all such proteins (Ross, E. and Schatz, G. (1976) J. Biol. Chem. 251, 1997-2004; Maccecchini, M. L., Rudin, Y., Blobel, G. and Schatz, G. (1979) Proc. Nat'l Acad. Sci. U.S.A. 76, 343-347; T., Goodman, J. M. and Wickner, W. (1980) Proc. Nat'l Acad. Sci. U.S.A. 77, 4669-4673).